The use of diagnostic test devices such as dry devices to analyze components in a sample of human body fluid such as urine is well known. A diagnostic test strip is one type of dry device that may be used to analyze components in human body fluid. Diagnostic test strips are typically composed of one or more pads of paper attached to a plastic carrier which serves as a handle.
With diagnostic test strips, a reagent such as an organic molecule is applied to or absorbed into an absorbent material such as paper by dipping the absorbent material into one or more solvent systems containing one or more reagents. Upon dipping the absorbent material into the solvent system, the reagent contained in the solvent system becomes absorbed within or integrated into the fibers of the absorbent material. Once the reagent has been applied to the absorbent material, the absorbent material is dried and assembled into a test strip.
The dried test strip containing the reagent can then be used to test for the presence of an analyte(s) in test samples. Test samples are typically human urine or other biological fluids. Test strips are frequently used to detect proteins in protein assays also referred to as total protein tests or proteinuria tests or to test for the presence of particular proteins in conditions such as albuminuria. Examples of analytes which are tested include, but are not limited to, proteins, hormones, drugs, metabolites, glucose, protons (i.e., for pH), ions (i.e., specific gravity), and blood cells. To test for the presence of an analyte(s), the test strip is dipped into the test sample at which point the reagent in the test strip participates in a reaction sequence with a particular analyte(s) in the test sample. Upon detecting the presence of a particular analyte(s) in the test sample, the reagent in the test strip responds with an instrumentally or visually detectable signal to the user such as a change in color.
In order for a reagent to become applied to the absorbent material of a diagnostic test device such as a test strip, the reagent needs to be soluble in the solvent system used to apply the reagent to the absorbent material. The more soluble the reagent is in this solvent system, the more reagent becomes applied to the absorbent material and the richer the color obtained on the absorbent material. A high concentration of reagent in the absorbent material often allows a rich color indication.
In order for a reagent which has been absorbed into the absorbent material of a diagnostic test device such as a test strip to be used to detect an analyte(s) in a test sample, the reagent that is trapped within the fibers of the absorbent material typically needs to be sufficiently soluble to be available to interact with the analyte(s) in the test sample. That is, the reagent that has been absorbed into the absorbent material typically needs to be soluble in the final test strip environment (i.e., the test sample) for solution chemistry to take place and to give an indication that a particular analyte(s) is present in the test sample. However, high solubility of the reagent in the test sample may be undesirable in some systems as problems such as reagent migration between multiple pads on the test strip can occur. An optimal system would allow assay designers to control the degree of reagent solubility in the test sample.
The organic reagents which are typically applied to the absorbent material of diagnostic test devices are either soluble in aqueous solvents with no solubility or only limited solubility in organic solvents or are soluble in organic solvents with no solubility or only limited solubility in aqueous solvents. In other words, many of these organic reagents have no transolubility or only limited transolubility in organic and aqueous solvents. The lack of transolubility of these reagents in organic and aqueous solvents has made the application of certain reagents to diagnostic test devices and the use of the diagnostic test devices containing these reagents difficult.
To illustrate, a solubility problem arises where an organic reagent must be applied to the absorbent material of diagnostic test devices using an organic solvent because the reagent is readily soluble only in organic solvents, but the reagent must subsequently be used to detect the presence of proteins in an aqueous test sample (i.e., urine) in which the reagent has no solubility or only limited solubility. For example, pyrogallol red is a dye commonly used in diagnostic protein tests that is soluble in organic solvents such as methanol but has no solubility or only limited solubility in aqueous solvents such as water or urine. One challenge facing assay designers has been to find a way to apply a reagent such as pyrogallol red to test strips via an aqueous solvent and to then use the reagent to detect the presence of proteins in an aqueous test sample in which the reagent has controlled solubility.
One method that has been used to dissolve organic reagents having limited transolubility in organic and aqueous solvents has been to increase the alkalinity of the aqueous solvent. This approach has been used to dissolve pyrogallol red dye in aqueous solvents and apply the dye via the aqueous solvent to the absorbent material such as the test paper. Although pyrogallol red may be applied to a diagnostic test device such as a test strip via an aqueous solvent using this approach, the strongly alkaline aqueous solvent needed to dissolve the pyrogallol red causes an unacceptable variation in the color of the test paper, rendering the paper unusable in diagnostic tests.
Another method used to dissolve organic reagents having limited transolubility in organic and aqueous solvents has been to add water miscible cosolvents, such as alcohols, to the solvent system. This approach also has shortcomings. The addition of a water miscible cosolvent may increase the solubility of one component in the solvent system but may simultaneously decrease the solubility of other component(s). In addition, the amount and type of water miscible cosolvent typically requires careful selection, often through a trial and error process, to increase the solubility of the organic reagent while maintaining the solubility of the water soluble components.
Because different reagents have varying degrees of solubility in the solvents used to apply reagents to the absorbent materials of diagnostic test devices such as test papers, it is sometimes necessary to use a multiple-dip or multiple-step application process. For example, a multiple-dip application process may be used where the desired reagents are reactive with each other or with one or more components contained in one of the solvent systems. Another challenge facing assay designers has been to find a way to control the transolubility of a reagent(s) where a multiple dip application process is involved. For example, in a two-dip application process, a reagent may be applied to a test paper using an aqueous solvent as the first dip solution, yet it is desirable that the reagent remain insoluble in the second, organic dip solution to prevent leaching of the reagent into the second dip solution that is being applied to the test paper.
For the foregoing reasons, there exists a need for a method to apply reagents having limited transolubility in organic solvents, aqueous solvents, and mixtures thereof to diagnostic test devices such as diagnostic test strips via organic solvents, aqueous solvents, and mixtures thereof for subsequent use in detecting the presence of an analyte(s) in test samples such as aqueous test samples. There also exists a need for a method to apply reagents having limited transolubility in organic solvents, aqueous solvents, and mixtures thereof to diagnostic test devices via organic solvents, aqueous solvents, and mixtures thereof (a) without adding, or with reduced need to add, cosolvents which affect the solubility of other components in the solvent and (b) without subjecting the reagent to harsh chemical conditions to dissolve the reagent in the desired solvent. A need also exists for a method to control the transolubility of a reagent(s) where a multiple dip application process is involved without side effects such as leaching of the reagent into subsequent dip solutions. There is also a need for a method to apply reagents to diagnostic test devices which allows assay designers to generally control or choose the degree of reagent solubility in the test sample. There also exists a need for diagnostic test strips which can be prepared without the above noted shortcomings and with the above noted advantages.